1.Role of Membrane Interactions on the Mechanism of alpha-Synuclein Amyloid Formation[unreadable] [unreadable] Understanding the environmental factors affecting the aggregation of alpha-synuclein (alpha-syn) is of great importance because the accumulation and deposit of alpha-syn are intimately connected to Parkinsons disease (PD) etiology. In the current study, synthetic phospholipid vesicles are employed as membrane mimics to investigate the mechanism of bilayer induced fibril formation. To monitor alpha-syn site specific interactions during the membrane binding process, four single fluorescent tryptophan residues were introduced at positions W4, W39, W94, W125. First, circular dichroism spectroscopy was employed to ensure overall membrane binding was conserved in the fluorescent mutants. Characteristic of all proteins studied, a saturable transition from an unfolded to a highly alpha-helical conformation was observed with a clear isodichroic point consistent with a two state equilibrium. The binding curves are adequately described by a sigmoidal function with midpoint transitions in the range of 120-160 lipid:protein ratio and comparable saturation limits at lipid:protein ratio >300. The differences in the midpoints likely reflect the uncertainty in the lipid concentration rather than the incorporation of the Trp fluorophore.[unreadable] [unreadable] Site specific environmental changes induced by increased addition of vesicles were monitored by steady state tryptophan fluorescence. The greatest changes in the emission spectrum upon lipid addition were observed for W4 with a pronounced blue shift (348 to 325 nm) and quantum yield increase. These spectral features highlight the role of the N terminal domain in lipid binding and in particular, the sensitivity of the indole fluorophore to the hydrophobic bilayer environment. In contrast, W39 located in the putative loop region of the micelle bound structure, exhibits only a modest blue shift and little quantum yield changes as a function of vesicles added suggesting that this sidechain is not necessarily direct contact with the membrane. Interestingly, a significant quantum yield increase also was observed for the C-terminal W94 side chain while, W125 was completely insensitive to the presence of the vesicles.[unreadable] [unreadable] These preliminary results clearly demonstrate that the N-terminus preferentially binds to the lipid bilayer. Furthermore, the incorporation of the fluorophores has minimal affects on alpha-synucleins physiological behavior of membrane binding. Interestingly, the lipid binding curves obtained using Trp fluorescence (spectral shift, quantum yield and average fluorescence lifetime) have lower lipid:protein midpoint transitions. The Trp spectroscopic changes suggest that initial alpha-syn-lipid interactions do not have concomitant secondary structural rearrangement. Current work is focused on gaining further insight into these site specific protein-lipid interactions using time resolved fluorescence measurements and isolated synaptic vesicle.[unreadable] [unreadable] 2. Copper(II) Binding to alpha-Synuclein[unreadable] [unreadable] Although recent work points to a genetic component to PD involving the accumulation and deposit of a neuronal protein, alpha-synuclein, the sporadic form of the disease is far more common and possibly connected to environmental factors that promote oxidative stress and aberrant redox-active metal metabolism. For example, selective accumulation of fibrils in dopaminergic neurons in PD has been attributed to the presence of easily oxidizable catechols that stimulate protein cross-links, as well as to increased iron concentration in Lewy bodies and copper in cerebrospinal fluid of PD patients. Furthermore, metal-enhanced oxidative oligomerization has been observed for alpha-synuclein in vitro, and, specific metal-protein interactions have been proposed to be critical in other neurodegenerative diseases involving amyloidogenic biomolecules such as amyloid beta-peptide (Alzheimers disease), prion protein (spongiform encephalopathies), and superoxide dismutase (amyotrophic lateral sclerosis). A difficult issue to resolve is whether metal ions perturb protein structures and thereby alter functions, or whether metal-protein complexes directly participate in the production of reactive oxygen species, or whether both mechanisms are at work.[unreadable] [unreadable] We have employed tryptophan fluorescence measurements to probe Cu(II)-protein binding in four mutants containing single W substitutions (F4W, Y39W, F94W, and Y125W). Upon addition of Cu(II), the tryptophan fluorescence is quenched site-specifically. The fluorophore at position 4 is the most strongly quenched (>80% at Cu(II) = protein) while W39 exhibits minimal quenching until more than one equivalent of Cu(II) has been added. Even though the participation of the N-terminal amino group and residues in the vicinity of 3-9 have been implicated in Cu(II) binding, our observation was somewhat unexpected, because H50 was identified previously as the primary Cu(II) ligand with the strongest affinity. Furthermore, neither W94 nor W125 reporters in the highly acidic (15 carboxylates) C-terminal region is sensitive to the presence of Cu(II) under these solution conditions.[unreadable] [unreadable] We examined the F4W protein in greater depth to probe binding in the N-terminal region because W4 is most sensitive to the presence of Cu(II). In addition, we replaced H50 with a serine residue (F4W/H50S variant) to assess the role, if any, of imidazole ligation to Cu(II). Upon copper(II) addition, tryptophan fluorescence is dramatically quenched, owing to direct Cu(II)-protein interactions. Titration curves for the two proteins (F4W and F4W/H50S) clearly demonstrate that one copper ion binds per protein, with a dissociation constant of <5 micormolar.[unreadable] [unreadable] To obtain reliable values for Cu-syn dissociation constants, we measured tryptophan excited-state decay kinetics at submicromolar protein concentrations (alpha-syn = 100 nM). We can fit the titration curves derived from the average lifetime data using a single-site binding model (F4W, Kd = 100(50) nM and F4W/H50S, Kd = 110(50) nM). The similarity in binding constants for the F4W and F4W/H50S proteins clearly indicates that the high-affinity site does not involve the H50 imidazole group, an observation that is consistent with recent NMR measurements on an H50A mutant.[unreadable] [unreadable] The high affinity N-terminal Cu(II)-binding site revealed by W4 fluorescence quenching does not involve H50 nor global polypeptide rearrangements that bring W39, W94, or W125 into proximity. We suggest that the tight binding involves the alpha-amino group of M1 and the carboxylate group of D2, along with deprotonated amides and carbonyl groups from the peptide backbone, consistent with previous work on synthetic N-terminal fragments, and similar to the structurally characterized Cu(II) site in the octarepeat domain of the prion protein. The details of intracellular copper-ion homeostasis are not well understood, so it is difficult to assess the importance of Cu(II)-alpha-syn binding in the natural function of the protein, or the disease state with which it is associated. [unreadable] Our experiments demonstrate that tryptophan fluorescence is a sensitive indicator of copper(II) binding to alpha-synuclein. Because we can make measurements at extremely low protein concentrations, we are able to extract reliable dissociation constants for high affinity binding sites. With this approach, we can determine how different alpha-syn subpopulations interact with other biomolecules and compounds that are implicated in Parkinsons disease.